Microbial Diversity and the Quality of Roof-harvested Rainwater
C.A. Evans, P.J. Coombes, R.H. Dunstan, T.L. Harrison, A. Martin, K. Pigott, J.N. Harris
School of Environmental and Life Sciences University of Newcastle Australia AreAre nonnon--pathogenicpathogenic bacteriabacteria significant?significant?
1. The ‘treatment train’ effect • Apparent improvements to the microbial and chemical quality of water with passage through the collection system. Contaminated run-off
‘Treatment train’
INLET
Improved water quality OUTLET AreAre nonnon--pathogenicpathogenic bacteriabacteria significant?significant?
1. The ‘treatment train’ effect • Apparent improvements to the microbial and chemical quality of water with passage through the collection system.
2. Environmental bacteria comprise majority of organisms present in rainwater tanks. •HPC >>> coliform counts - (orders of magnitude) • Bacterial composition influenced by wind patterns WindWind direction/velocitydirection/velocity && HPCHPC
6000 (r2 = 0.98, p<0.001) 5000 )
L 4000 m / u
f N c 3000 C ( S 2000 2 HP (r = 0.85, p<0.03)
1000
0 02468 Wind velocity (m/sec) Contaminated run-off
‘Treatment train’
INLET Biofilm formation
Nutrient cycling
Chemical remediation
Competitive exclusion of pathogens Improved water quality OUTLET OverviewOverview
1. Assessment of microbial diversity in 2 rainwater tanks. • Impact of hot water systems • Assessment of coliforms
2. Biofilm pilot studies • Biofilm development and the treatment train • In-vitro examination of Pb2+ uptake MicrobialMicrobial DiversityDiversity ofof 22 RainwaterRainwater TanksTanks
• Urban tank – Located in Newcastle (160kM Nth of Sydney) – Duel 2.2kL galvanized iron tanks – Mains water top-up system – Hot water service set at 60°C
• Rural tank – Located at Gulgong (250kM inland of Newcastle) – 10kL ‘aquaplate’ tank – Hot water service set at 50°C. Table 1: Plate count and diversity summary
Tank Urban Rural
Sample type Cold Hot Cold Hot
Total no. of species identified 64 20 49 39
mean no. species/sample 23 ± 3.4 8 ± 1.2 16 ± 2.3 14 ± 4.3
Mean plate count (cfu/mL) 366 ± 153 17 ± 2.6 2263 ± 1070 94 ± 31 Table 1: Plate count and diversity summary
Tank Urban Rural
Sample type Cold Hot Cold Hot
Total no. of species identified 64 20 49 39
mean no. species/sample 23 ± 3.4 8 ± 1.2 16 ± 2.3 14 ± 4.3
Mean plate count (cfu/mL) 366 ± 153 17 ± 2.6 2263 ± 1070 94 ± 31 Table 1: Plate count and diversity summary
Tank Urban Rural
Sample type Cold Hot Cold Hot
Total no. of species identified 64 20 49 39
mean no. species/sample 23 ± 3.4 8 ± 1.2 16 ± 2.3 14 ± 4.3
Mean plate count (cfu/mL) 366 ± 153 17 ± 2.6 2263 ± 1070 94 ± 31 Table 1: Plate count and diversity summary
Tank Urban Rural
Sample type Cold Hot Cold Hot
Total no. of species identified 64 20 49 39
mean no. species/sample 23 ± 3.4 8 ± 1.2 16 ± 2.3 14 ± 4.3
Mean plate count (cfu/mL) 366 ± 153 17 ± 2.6 2263 ± 1070 94 ± 31 Table 1: Plate count and diversity summary
Tank Urban Rural
Sample type Cold Hot Cold Hot
Total no. of species identified 64 20 49 39
mean no. species/sample 23 ± 3.4 8 ± 1.2 16 ± 2.3 14 ± 4.3
Mean plate count (cfu/mL) 366 ± 153 17 ± 2.6 2263 ± 1070 94 ± 31 Table 1: Plate count and diversity summary
Tank Urban Rural
Sample type Cold Hot Cold Hot
Total no. of species identified 64 20 49 39
mean no. species/sample 23 ± 3.4 8 ± 1.2 16 ± 2.3 14 ± 4.3
Mean plate count (cfu/mL) 366 ± 153 17 ± 2.6 2263 ± 1070 94 ± 31 Table 1: Plate count and diversity summary
Tank Urban Rural
Sample type Cold Hot Cold Hot
Total no. of species identified 64 20 49 39
mean no. species/sample 23 ± 3.4 8 ± 1.2 16 ± 2.3 14 ± 4.3
Mean plate count (cfu/mL) 366 ± 153 17 ± 2.6 2263 ± 1070 94 ± 31 Fig. 2: % composition of gram negative and gram positive bacteria in samples from rainwater tanks.
100% 90% 80% n
io 70% it
s 60% o
p G+ve 50% m
o G-ve 40% c
% 30% 20% 10% 0% Ncle(cold) Gulg(cold) Ncle(hot) Gulg(hot) Sample type Fig. 1: Profiles of the Newcastle and Gulgong tanks based on average counts of most abundant genera present.
450 400 )
l 350 300 250 Gu lg. 200 Nc le 150
Avge count (cfu/m 100 50 0 . . p. . . i p. p. sp p p. p p ed s s s s fi ng s s a s ti um ia s us s nti Fu um ona a lftia ri in ll ll na rr e te ci ide iri o D c rs n dom Se a Ye Ba U sp om ob ba ng eu n er hi Ps H nthi Sp Ja Faecal indicator presence and abundance relative to total plate count
Sample type COLD HOT
Presence Avge % of Presence Avge % of (% of samples) plate count (% of samples) plate count
E.coli 50 0.06 ± 0.03 0 0
Enterococci 23 0.05 ± 0.03 28 0.62 ± 0.41
Faecal col. 77 0.52 ± 0.27 28 0.62 ± 0.41
Total col. 100 3.77 ± 2.08 43 3.36 ± 2.21 BiofilmsBiofilms andand thethe treatmenttreatment traintrain
Location: Newcastle on east coast of Australia (approx. 160kM Nth of Sydney)
Tank Biofilm development Comment
A (G.I.) Advanced heavy slime layer with extensive clumping
B (Plastic) Moderate uniform slime layer around interior Table 3: Variation in bacterial counts between the tank water column and tap outlet of tanks A and B.
Tank Sample type Bacterial count E.coli HPC (cfu/100ml) (cfu/mL)
A Tank water column 48 610 Tap outlet 29 390
Improvement 34% 36%
B Tank water column 70 200 Tap outlet 60 160
Improvement 14% 20% Table 3: Variation in bacterial counts between the tank water column and tap outlet of 2 tanks.
Tank Sample type Bacterial count E.coli HPC (cfu/100ml) (cfu/mL)
A Tank water column 48 610 Tap outlet 29 390
Improvement 34% 36%
B Tank water column 70 200 Tap outlet 60 160
Improvement 14% 20% PbPb2+ uptakeuptake byby inin--vitrovitro cultivatedcultivated biofilmbiofilm
• E.coli biofilms cultivated in wells of perspex tissue culture plates for 72 hours.
• Incubation broth replaced with sterile milli-Q water spiked with Pb2+ (100ppb).
•Pb2+ determined by ICP-MS at time = 0, 5, 10 and 20 hours. Pb2+ uptake by in-vitro cultivated biofilm
110
100 90 ppb) ( . 80 Biofilm nc o 70 Control c d a 60 Le 50
40 0 5 10 15 20 25 Time (hrs) ConclusionsConclusions
• Rainwater tanks harbour a wide diversity of microbial species.
• Species composition may vary considerably between tanks at different locations.
• Species composition varies temporally at any given site.
• Minimal evidence of faecal contamination.
• Hot water systems deliver water of quality suitable for bathing.
• Biofilms have capacity to chemically remediate water. Phylum Class Order Family Genus Sphingomonadales Sphingomonadaceae Novosphingobium Bradyrhizobiaceae Afipia Porphyrobacter α Rhizobiales Sphingomonas Methylobacteriaceae Methylobacterium Caulobacterales Caulobacteraceae Caulobacter Burkholderiaceae Burkholderia Comomonadaceae Acidovorax Comomonas Burkholderiales Oxalobacteriaceae Duganella Delftia Massilia Polaromonas Naxibacter Variovorax Herbaspirillum Ralstoniaceae Janthinobacterium Ralstonia Proteobacteria β Nesseriales Nesseriaceae Aquaspirillum Aquitalea Chromobacterium Rhodocyclales Rhodocyclaceae Dechloromonas Iodobacter Microvirgula Enterobacteriales Enterobacteriaceae Citrobacter Enterobacter Aeromonadales Aeromonadaceae Aeromonas Erwinia Escherichia Chromatiales Chromatiaceae Rheinheimera Hafnia γ Klebsiella Moraxellaceae Acinetobacter Leclercia Pseudomonadales Moraxella Obesumbacterium Pseudomonadaceae Pseudomonas Pantoea Proteus Xanthomonadales Xanthomonadaceae Luteibacter Rahnella Stenotrophomonas Raoultella Pseudoxanthomonas Serratia Clostridium Clostridia Clostridiales Clostridiaceae Yersinia Bacillaceae Bacillus Exiguobacterium Listeriaceae Listeria Bacillales Planococcaceae Planococcus Firmicutes Planomicrobium Paenibacillaceae Brevibacillus Sporasarcina Paenibacillus Staphylococcaceae Staphylococcus Bacilli Aerococcaceae Aerococcus Carnobacteriaceae Carnobacterium Desemzia Corynebacteriaceae Corynebacterium Lactobacillales Enterococcaceae Enterococcus Microbacteriaceae Frigoribacterium Microbacterium Streptococcaceae Lactococcus
Micrococcaceae Arthrobacter Leucobacter Gordoniaceae Gordonia Micrococcus Actinobacteria Actinobacteria Actinomycetales Mycobacteriaceae Mycobacterium Nocardiaceae Rhodococcus Streptomycetaceae Streptomyces
Flavobacteria Flavobacteroidales Flavobacteriaceae Chryseobacterium Flavobacterium Bacteroidetes Flexibacteriaceae Flectobacillus Sphingobacteria Sphingobacteriales Hymenobacter Sphingobacteriaceae Sphingobacterium